The long-term goals of our research are to define structure-function relationships of the Na,K-ATPase and to understand the tissue and developmental specific expression of the a subunit isoforms. The Na,K-ATPase is an integral membrane enzyme found in cells of higher organisms and is responsible for the ATP dependent transport of Na4+ and K+ ions across the cell membrane. cDNA and genomic cloning have described three isoforms of the a subunit and two isoforms of the beta subunit. These isoforms are expressed differentially in various tissues and during development. The availability of cDNA clones has allowed site-specific mutagenesis to be carried out to study structure-function relationships of the enzyme including identification of regions involved in sensitivity to cardiac glycosides. Our present proposal is a continuation of work already in progress in our laboratory and our specific aims are as follows: We will continue our studies directed toward defining the cardiac glycoside binding site of Na,K-ATPase using a powerful positive-negative selection system. Studies have shown that the first extracellular domain is involved and we are now using both site-specific and random mutagenesis to identify other regions. Studies are also underway to identify enzymatic differences between the alphal, alpha2 and alpha3 isoforms and to determine domains responsible for these differences. Chimeric enzymes produced by combining portions of the cDNAs of the isoforms are being employed for these studies. Site-- specific mutagenesis is being utilized to identify amino acids Involved in Na+ and/or K+ binding taking advantage of a positive-negative selection procedure. It is also hoped that a system can be developed which will overproduce the enzyme to allow additional studies of structure-function to be carried out. The regulation of isoform expression will also be pursued. We are particularly interested in identifying cis elements required for the up regulation of the alpha2 isoform gene when C2Cl2 skeletal muscle cell myoblasts fuse to form myotubes. Once cis elements are identified, trans acting factors which interact with these elements will be defined and characterized. Finally, the elements responsible for tissue- and developmental-specific expression will be studied using transgenic mouse assays. The alpha2 promoter attached to a CAT reporter gene has already been shown to be appropriately expressed in transgenic mice. Our studies represent a mix of gene regulation and structure-function studies which should begin to provide details on the mechanism of cation translocation, the functional characteristics of the alpha isoforms and the basis for differential expression of the a isoform in various tissues and during development.